GSM8084286: Monolayer 4; Homo sapiens; RNA-Seq
Spheroids were spun down at 500g at 4C for 5 min and the PBS/1% BSA solution was carefully removed. For downstream analyses, the spheroids were recovered from Matrigel in 1 ml of cell recovery solution (354253, Corning) for 30 min on ice with occasional mixing. Spheroids were washed once with 1 ml of cold PBS and lysed in TriPure Isolation Reagent at 700 L/sample (11667165001, Sigma). Chloroform was added at a volume of 140 uL per sample, and the tubes were shaken vigorously for 15 seconds and allowed to stand at room temperature for 10-15 minutes. Samples were centrifuged at 12,000 g for 15 minutes at 48C, after which 350 mL of the upper aqueous phase was collected and mixed at a 1:1 ratio with 100% ethanol. Further isolation was performed by using the Quick-RNA MicroPrep Kit (Zymo Research, Irvine, Calif, catalog no. R1051) per the manufacturer's instructions. The RNA concentration was measured by Nanodrop, and the RNA integrity number was determined by the Gene Expression Core at CCHMC by using the Agilent 2100 Bioanalyzer. Standard Illumina protocol